Histones are known to be modified by covalent binding of the vitamin biotin Histone biotinylation is unique among histone modifications in that a metabolic co-factor also functions as a chromatin mark. We have identified ten distinct biotinylation sites in histones H2A, H3, and H4. Additional biotinylation sites are known to exist but have only been tentatively identified. Biotinylation of histones is mediated by holocarboxylase synthetase (HCS) and has important biological functions, e.g., in the repression of retrotransposons, in heterochromatin structures and DNA repair, and in gene regulation. The elucidation of the full spectrum of biological functions of histone biotinylation is currently slowed by our incomplete understanding of histone biotinylation sites, the lack of availability of antibodies to some biotinylation sites, and our lack of understanding of the crosstalk between histone biotinylation and other epigenetic marks. Here, we propose to overcome these obstacles by taking advantage of a unique combination of investigators with expertise in diverse areas of research: a protein biochemist who also is the head of a mass spec laboratory (Chang), a geneticist with extensive expertise in epigenetic mechanisms of gene regulation (Eissenberg), and a nutritionist who discovered histone biotinylation and developed many unique tools in this field (Zempleni). The long-term objective of this project is to identify pathways by which histone biotinylation maintains genome stability. Specific aims: (1) In aim 1, we will identify all naturally occurring biotinylation sites in all histones and variants in living cells by using LC/MS/MS. In addition, we will generate antibodies to novel biotinylation sites. These antibodies will be essential for use in future studies to determine the biological importance of histone biotinylation. (2) In aim 2 we will test the hypothesis that biotinylation co-exists with other modification marks on the same histone molecule in humans. We will generate antibodies to selected examples of histones with multiple modifications. First insights into biological functions will be generated by mapping these modified histone isoforms in Drosophila polytene chromosomes. (3) Ongoing projects in our laboratories suggest that HCS interacts with H3 K9-methyl transferases and that K12-biotinylated histone H4 co-localizes with K9- dimethylated histone H3. Here we will test the hypothesis that HCS knockdown in human cells and flies decreases the abundance of K9Me3H3 at selected loci, and increases the abundance of K4Me3H3 at these loci. We will also test the hypothesis that biotinylation of histones decreases in response to knockdown, mutation, and/or overexpression of K9-methyl transferases. Most of these mutant and transgenic organisms are in our laboratories or freely available. PUBLIC HEALTH RELEVANCE: Biotinylation of histones is a unique epigenetic mark because it depends on the dietary intake of the essential vitamin biotin. Biotin deficiency is prevalent among Americans, and moderate biotin deficiency has been observed in up to 50% of pregnant women. Previous studies suggest that biotinylation of histones plays a critical role in the repression of retrotransposons, thereby decreasing the incidence of retrotranspositions, chromosomal abnormalities, and probably cancer risk.